Iodinated radiocontrast media are used to enhance the visualization of blood vessels and internal organs for a wide range of diagnostic and/or interventional procedures, including (but not limited to) angiography, urography, pyelography, arthrography, cholangiography, diskography, myelography, contrast-enhanced computer tomography, and cerebral ventriculography. Iodinated radiocontrast media are usually classified as ionic or nonionic. The nonionic radiocontrast media are organic compounds containing covalently bound iodine and are either monomeric or dimeric molecules with three or six atoms of iodine, respectively. Ionic iodinated radiocontrast media were developed before nonionic iodinated radiocontrast media. Both types of iodine-based contrast media are water soluble. Iodinated radiocontrast media are relatively safe drugs, but can cause serious anaphylactic reactions or contrast-induced nephropathy in some patients. The incidence of contrast-induced nephropathy in the general hospital population is low (1-2%), but can be above 50% in some hospital subpopulations, such as elderly diabetic patients with chronic kidney disease. Nonionic iodinated radiocontrast media are less nephrotoxic than ionic iodinated radiocontrast media. Contrast-induced nephropathy is the third leading cause of iatrogenic kidney failure (Thomsen et al., Acta Radiol 49:646-657, 2008). The risk factors for development of contrast-induced nephropathy include pre-existing kidney diseases, diabetes mellitus, hypertension, cardiovascular diseases, sickle cell anemia, gout flares, multiple myeloma, hypoalbuminemia, hypovolemia, and dehydration. Intravenous administration of iodinated radiocontrast media is less nephrotoxic than administration of the media into either the renal artery or the aorta proximal to the origin of the renal artery (Katzberg & Barrett, Radiology 243:622-628, 2007).
The iodinated radiocontrast media iodixanol (Visipaque), iohexol (Omnipaque), iopamidol (Isovue), iopentol (Imagopaque), iopromide (Ultravist), ioversol (Optiray), ioxilan (Oxilan), iothalamate (Conray), ioxaglate (Hexabrix), metrizamide (Amipaque), metrizoate (Isopaque), and sodium diatrizoate and meglumine diatrizoate (Urografin) have been approved by the U.S. Food and Drug Administration (FDA) for numerous diagnostic and/or interventional procedures. The iodinated radiocontrast media iobitridol (Xenetix), iodipamide (Bilignost), iomeprol (lomeron), iotrolan (Isovist), and ioxithalamate (Telebrix) are used in the European Union.
The development of therapeutics for the treatment, management or prevention of injury to the kidney of humans or other mammals caused by one or more iodinated radiocontrast media has been hampered by the lack of a sensitive noninvasive method for detecting the earliest signs of kidney injury. Acute kidney injury in the clinic is usually diagnosed by determining the concentration of creatinine in serum. Contrast-induced nephropathy is usually defined as an increase in serum creatinine of more than 25% or an increase of more than 0.5 mg/dl within three days after the intravascular administration of contrast media that cannot be explain by other known causes. However, serum creatinine is an insensitive and nonspecific indicator of impaired kidney function. A large reduction in the glomerular filtration rate can occur before there is a detectable increase in serum creatinine. Urinary levels of kidney injury molecule 1 (KIM-1) and netrin-1 have been claimed to be more sensitive biomarkers for the earliest signs of kidney injury than serum levels of creatinine (Waanders et al., J Pathol 220:7-16, 2010; Ramesh et al., Transplant Proc 42:1519-1522, 2010). Nogo-B (reticulon 4B) is up-regulated in the proximal tubules of the mouse kidney following unilateral ureteral obstruction and ischemia/reperfusion, and Nogo-B mRNA levels have been suggested to be a sensitive indicator for diverse forms of renal proximal tubule injury (Marin et al., Am J Pathol 177:2765-2773, 2010).
The antioxidants N-acetylcysteine, vitamin E (α-tocopherol) and ascorbic acid have been used as adjunctive cytoprotective agents with iodinated radiocontrast media in small clinical trials without any consistent clear cut evidence of benefit. None of these antioxidants acts via G-protein-coupled receptors and classical signal transduction pathways, and any of these antioxidants could easily be used in combination with PACAP-based therapeutics. Vasodilators and vasoconstrictor antagonists, including fenoldopam, SB 209670, atrial natriuretic peptide, theophylline, and aminophylline, have also been used as adjunctive cytoprotective agents with iodinated radiocontrast media in small clinical trials without any consistent clear cut evidence of benefit. The intravenous administration of isotonic fluids is the only generally accepted strategy to reduce the incidence of contrast-induced nephropathy. There are currently no drugs that are approved by the U.S. FDA specifically for use as cytoprotective adjunctive agents with iodinated radiocontrast media.
Pituitary adenylate cyclase-activating polypeptide (PACAP) was isolated from ovine (sheep) hypothalami based on its ability to stimulate adenylate cyclase activity in rat anterior pituitary cell cultures (Miyata et al., Biochem Biophys Res Commun 164:567-574, 1989). PACAP exists as two α-amidated peptides with 38 (PACAP38; SEQ ID NO:1) or 27 (PACAP27; SEQ ID NO:2) amino acids. Both peptides have the same N-terminal 27 amino acids and are synthesized from the same prohormone. The sequence of PACAP38 is identical in all mammals and differs from the reptilian, avian and amphibian orthologs by only one amino acid (Vaudry et al., Pharmacol Rev 52:269-324, 2000; Valiante et al., Brain Res 1127:66-75, 2007; Vaudry et al., Pharmacol Rev 61:283-357, 2009). PACAP is a member of the secretin/vasoactive intestinal peptide (VIP)/growth hormone-releasing hormone (GHRH) family, and PACAP27 has 68% sequence identity with VIP (SEQ ID NO:3). PACAP is most abundant in the brain and testis, but there are significant levels in other organs, including the adrenals, thymus, spleen, lymph nodes, and duodenal mucosa (Vaudry et al., Pharmacol Rev 52:269-324, 2000; Vaudry et al., Pharmacol Rev 61:283-357, 2009). The amount of PACAP in the kidney is relatively low (Arimura et al., Endocrinology 129:2787-2789, 1991). PACAP is synthesized as a preprohormone and is processed mainly by prohormone convertase 1, prohormone convertase 2 and prohormone convertase 4 (Li et al., Neuroendocrinology 69:217-226, 1999; Li et al., Endocrinology 141:3723-3730, 2000). The half-life of [125I]-PACAP38 in the bloodstream of rats following intravenous injection is 5-6 minutes (Banks et al., J Pharmacol Exp Ther 267:690-696, 1993). Members of the secretin/VIP/GHRH family are degraded in plasma mainly by aminodipeptidases, especially dipeptidyl peptidase IV (Zhu et al., J Biol Chem 278:22418-2223, 2003).
A PACAP-specific receptor, designated as the PAC1 receptor, has been cloned from several vertebrate species (Arimura, Jpn J Physiol 48:301-331, 1998; Vaudry et al., Pharmacol Rev 52:269-324, 2000; Vaudry et al., Pharmacol Rev 61:283-357, 2009). It is a G-protein-coupled receptor with seven putative membrane-spanning domains and belongs to a family of glycoprotein receptors that are coupled to multiple signal transduction pathways (Segre & Goldring, Trends Endocrinol Metab 4:309-314, 1993). PACAP binds not only to the PAC1 receptor with a high affinity, but it also binds to the VIP1 (VPAC1) and VIP2 (VPAC2) receptors with an affinity comparable to or greater than VIP. On the other hand, VIP binds to the PAC1 receptor with an affinity 100-1,000 times less than PACAP (Arimura, Jpn J Physiol 48:301-331, 1998). At least 10 splice variants of the rat PAC1 receptor have been cloned and each variant is coupled to distinct combinations of signal transduction pathways (Vaudry et al., Pharmacol Rev 52:269-324, 2000; Vaudry et al., Pharmacol Rev 61:283-357, 2009). The “second” messengers include adenylate cyclase, phospholipase C, mitogen-activated protein (MAP) kinases, and calcium. PACAP/VIP receptor can be coupled to Gαs and/or Gαi/o in different types of cells. PACAP/VIP receptors are expressed in many different types of normal cells, including the catecholamine-containing cells in the adrenal medulla and the sympathetic ganglia; microglia, astrocytes and some types of neurons in the central nervous system; and T- and B-lymphocytes, macrophages and dendritic cells in the immune system (Vaudry et al., Pharmacol Rev 52:269-324, 2000; Vaudry et al., Pharmacol Rev 61:283-357, 2009). PACAP is a potent stimulator of catecholamine secretion from the adrenal medulla (Watanabe et al., Am J Physiol 269:E903-E909, 1995), but a potent inhibitor of the secretion of tumor necrosis factor-α (TNF-α), interleukin (IL)-6 and IL-12 from activated macrophages (Ganea & Delgado, Crit Rev Oral Biol Med 13:229-237, 2002). More pertinent to the present invention, PACAP has been reported to increase blood flow in the kidney (Nilsson, Eur J Pharmacol 253:17-25, 1994).
Although PACAP was isolated during a screen for novel hypophysiotropic factors, it soon became apparent that it is a pleiotropic peptide (Arimura, Jpn J Physiol 48:301-331, 1998; Vaudry et al., Pharmacol Rev 52:269-324, 2000). The extraordinarily potent neuroprotective/neurotrophic properties of PACAP were investigated by several laboratories shortly after its isolation. The cytoprotective effects of PACAP and VIP have been studied much more extensively in the nervous system than in any other major organ of the body. For example, PACAP prevented the neuronal death induced by gp120, the envelope glycoprotein of the human immunodeficiency virus (HIV), in rat hippocampal neuron/glia co-cultures. The dose-response curve was bimodal, with peaks at 10−13 M and 10−10 M (Arimura et al., Ann NY Acad Sci 739:228-243, 1994). The critical findings in this study have been confirmed by Kong et al. (Neuroscience 91:493-500, 1999), who used lipopolysaccharide as the neurotoxin in primary murine cortical neuron/glia co-cultures. The neuroprotective effect at 10−12 M was correlated with a significant reduction in the accumulation of nitrite in the culture medium. The neuroprotective effect of “low” (femtomolar) doses of PACAP in neuron/glia co-cultures was abolished by PD98059, a MAP kinase inhibitor, but the neuroprotective effect of “high” (nanomolar) doses of PACAP was not affected by PD98059 (Li et al., J Mol Neurosci 27:91-106, 2005). However, the neuroprotective effect of nanomolar doses of PACAP was abolished by Rp-cAMP, a protein kinase A inhibitor.
The drawbacks of using peptides for neuroprotection in the brain include their poor transport across the blood-brain barrier and their short half-life in the circulation after systemic administration. However, PACAP38 is transported from the blood to the brain via a saturable mechanism (Banks et al., J Pharmacol Exp Ther 267:690-696, 1993). Delayed systemic administration of PACAP has been shown to be neuroprotective in common in vivo preclinical models for both heart attack (Uchida et al., Brain Res 736:280-286, 1996) and stroke (Reglodi et al., Stroke 31:1411-1417, 2000). PACAP has also been shown by other laboratories to be neuroprotective in common in vivo preclinical models for other neurological diseases, including spinal cord injury (Chen & Tzeng, Neurosci Lett 384:117-121, 2005), Alzheimer's disease (Rat et al., FASEB J 25:3208-3218, 2011) and Parkinson's disease (Reglödi et al., Behav Brain Res 151:303-312, 2004).
The neuroprotective effects of low concentrations of PACAP in the nervous system are indirect and are probably mediated by at least four distinct mechanisms. (1) PACAP is a potent anti-inflammatory peptide. It has been shown to inhibit the induction of inducible nitric oxide synthase (iNOS) in activated macrophages, to inhibit the production of the pro-inflammatory cytokines TNF-α, IL-6 and IL-12 in activated macrophages, and to stimulate the production of the anti-inflammatory cytokine IL-10 in activated macrophages (Ganea & Delgado, Crit Rev Oral Biol Med 13:229-237, 2002). PACAP is also an extraordinarily potent “deactivator” of activated microglial cells (Kong et al., Neuroscience 91:493-500, 1999; Delgado et al., Glia 39:148-161, 2002), which are the resident macrophage-like cells in the nervous system. (2) Femtomolar (10−15 M) concentrations of PACAP increase the levels of the mRNA for activity-dependent neurotrophic factor in murine neuron/glia co-cultures (David et al., Society for Neuroscience [33rd Annual Meeting], New Orleans, La., #38.1 [Abstract], 2003). (3) Yang et al. (J Pharmacol Exp Ther 319:595-603, 2006) have shown that femtomolar concentrations of PACAP inhibit microglial NADPH oxidase activity and extracellular superoxide levels in mesencephalic neuron/glia co-cultures. (4) Figiel & Engele (J Neurosci 20:3596-3605, 2000) have reported that PACAP increased the expression of the glutamate transporters GLT-1 and GLAST and increased the activity of the glutamate metabolizing enzyme glutamine synthetase in astrocytes. These effects of PACAP would be expected to decrease glutamatergic neurotransmission.
The cytoprotective properties of PACAP and VIP have been studied far less extensively in the kidney than in the nervous system. PACAP has been shown to protect the kidney against injuries caused by ischemia/reperfusion (Riera et al., Transplantation 72:1217-1223, 2001; Szakaly et al., J Mol Neurosci 36:89-96, 2008; Li et al., Am J Nephrol 32:522-532, 2010), the commonly used aminoglycoside antibiotic gentamicin (Li et al., Regul Pept 145:24-32, 2008), light-chain immunoglobulin overload (Li et al., Regul Pept 145:24-32, 2008), and acute administration of cisplatin (Li et al., 2009, Li et al., J Mol Neurosci 43:58-66, 2011) and cyclosporine A (Khan et al., J Invest Med 59:793-802, 2011). Nephrotoxicity is usually the “dose-limiting” toxicity for gentamicin, cisplatin and cyclosporine A. The renoprotective effects of PACAP, like the neuroprotective effects of PACAP, are associated with suppression of innate immune responses (e.g., Li et al., J Mol Neurosci 43:58-66, 2011). PACAP has also been shown to inhibit the production of transforming growth factor (TGF)-β1 by macrophages (Sun et al., J Neuroimmunol 107:88-99, 2000).
PACAP-like peptides have been shown to inhibit the proliferation of most normal hematopoietic cells (e.g., Ottaway & Greenberg, J Immunol 132:417-423, 1984; Boudard & Bastide, J Neurosci Res 29:29-41, 1991; Tatsuno et al., Endocrinology 128:728-734, 1991; Trejter et al., Histol Histopathol 16:155-158, 2001).
Native PACAP has already been administered to healthy human volunteers by investigators in at least five different laboratories (Hammond et al., J Endocrinol 137:529-532, 1993; Chiodera et al., Neuroendocrinology 64:242-246, 1996; Filipsson et al., J Clin Endocrinol Metab 82:3093-3098, 1997; Doberer et al., Eur J Clin Invest 37:665-672, 2007; Murck et al., Am J Physiol 292:E853-E857, 2007) and to a patient with multiple myeloma under a U.S. FDA-approved single-patient protocol (Li et al., Peptides 28:1891-1895, 2007). The only untoward effect reported was a transient flushing.
The published literature indicates that PACAP-like peptides can protect neurons (neuroepithelial cells) against a very broad range of injuries, including ischemia/reperfusion injury. The published literature also indicates that PACAP-like peptides can protect renal epithelial cells against injury due to ischemia/reperfusion.
Whether PACAP-like peptides can protect renal epithelial cells against iodinated radiocontrast agents has not been previously investigated.
Citation or discussion of a reference herein shall not be construed as an admission that such reference is prior art to the present invention.